ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2003, p. 2674–2681 Vol. 47, No. 80066-4804/03/$08.00�0 DOI: 10.1128/AAC.47.8.2674–2681.2003Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Dinucleotide Analogues as Novel Inhibitors of RNA-DependentRNA Polymerase of Hepatitis C Virus

Weidong Zhong,* Haoyun An, Dinesh Barawkar, and Zhi HongDrug Discovery, Ribapharm, Inc., Costa Mesa, California 92626

Received 5 March 2003/Returned for modification 12 May 2003/Accepted 23 May 2003

Replication of hepatitis C virus (HCV) RNA is catalyzed by the virally encoded RNA-dependent RNA poly-merase NS5B. It is believed that the viral polymerase utilizes a de novo or primer-independent mechanism forinitiation of RNA synthesis. Our previous work has shown that dinucleotides were efficient initiation moleculesfor NS5B in vitro (W. Zhong, E. Ferrari, C. A. Lesburg, D. Maag, S. K. Ghosh, C. E. Cameron, J. Y. Lau, andZ. Hong, J. Virol. 74:9134-9143, 2000). In this study, we further demonstrated that dinucleotide analoguescould serve as inhibitors of de novo initiation of RNA synthesis directed by HCV NS5B. Both mononucleotide-and dinucleotide-initiated RNA syntheses were affected by dinucleotide analogues. The presence of the 5�-phosphate group in the dinucleotide compounds was required for efficient inhibition of de novo initiation.Optimal inhibitory activity also appeared to be dependent on the base-pairing potential between the com-pounds and the template terminal bases. Because the initiation process is a rate-limiting step in viral RNAreplication, inhibitors that interfere with the initiation process will have advantages in suppressing virusreplication. The use of dinucleotide analogues as inhibitor molecules to target viral replication initiationrepresents a novel approach to antiviral interference.

Hepatitis C virus (HCV) infection is an important publichealth problem worldwide and is recognized as the major causeof non-A, non-B hepatitis. It is estimated that HCV affects 4million people in the United States, 8 million people in Europeand Japan, and, collectively, 170 million people worldwide (22,24). Although HCV infection resolves in some cases, the virusestablishes chronic infection in up to 80% of the infectedindividuals and persists for decades. It is estimated that about20% of these infected individuals will go on to develop cirrho-sis, and 1 to 5% will develop liver failure and hepatocellularcarcinoma (23, 24, 26). Chronic hepatitis C is the leading causeof chronic liver disease and the leading indication for livertransplantation in the United States. The Centers for DiseaseControl and Prevention estimate that hepatitis C currently isresponsible for approximately 8,000 to 10,000 deaths in theUnited States annually. This number is projected to increasesignificantly over the next decade. Currently, there is no vac-cine for HCV infection due to the high degree of heterogeneityof this virus.

The objectives for the treatment of chronic hepatitis C are toachieve complete and sustained clearance of HCV RNA inserum and normalization of serum alanine aminotransferaselevels. In the absence of a prophylactic vaccine or a highlyspecific antiviral agent, treatment options for chronically in-fected individuals are limited. The current treatment optionsfor chronic hepatitis C include (pegalated) alpha interferon(IFN-�) monotherapy and (pegalated) IFN-�–ribavirin com-bination therapy, with sustained virological response rates ofbetween 10 and 60% (4, 7, 15–17, 20, 21). Clearly, more effec-tive and direct antiviral interventions are necessary for further

prevention and treatment of the life-threatening complicationscaused by HCV infection. Efforts to identify and develop high-ly specific and potent HCV inhibitors have intensified recently.Investigators have targeted all regions of the HCV genome andvirally encoded replication enzymes for potential therapeuticdiscovery.

HCV is a positive-strand RNA virus belonging to the familyFlaviviridae (3). This virus family also contains about 40 flavi-viruses that are associated with human diseases, including thedengue fever viruses, yellow fever viruses and Japanese en-cephalititis virus, as well as pestiviruses, whose infection ofdomesticated livestock can cause significant economic lossesworldwide. Like other RNA viruses, a virally encoded replica-tion enzyme, RNA-dependent RNA polymerase (RdRp), playsa central role in viral RNA replication of HCV and othermembers of the family Flaviviridae. In the case of HCV, thereplication protein is termed “NS5B” (nonstructural protein5B) (2, 5, 6, 12, 13). RdRp proteins are the key components ofthe viral replicase complexes and therefore serve as attractivetargets for antiviral development (1, 8, 9, 11).

It is generally believed that HCV RNA replication is initi-ated by NS5B RdRp via a de novo or primer-independentmechanism (10, 14, 18, 19, 25, 28). Our previous observationssuggest that dinucleotides can be used more efficiently as ini-tiation molecules by HCV NS5B than mononucleotides in thein vitro RNA replication assay (27). It is thus perceivable thatdinucleotide analogues may have potential to inhibit initiationof HCV RNA replication. Because the initiation process isconsidered to be the rate-limiting step in viral RNA replica-tion, inhibitors that interfere with the initiation process willhave advantages in suppressing virus replication. The use ofdinucleotide analogues as inhibitor molecules to target theinitiation step of viral RNA synthesis represents a novel ap-proach to antiviral interference.

* Corresponding author. Mailing address: Ribapharm, Inc., 3300Hyland Ave., Costa Mesa, CA 92626. Phone: (714) 427-6236, ext. 2201.Fax: (714) 668-3141. E-mail: wzhong@ribapharm.com.

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FIG. 1. Chemical synthesis schemes of dinucleotide analogues I (A) and II (B).

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FIG. 2. Inhibition of HCV NS5B-catalyzed de novo initiation of RNA synthesis by dinucleotide analogues. (A) Structures of dinucleotideanalogues I and II. (B) Inhibition of NS5B-directed de novo initiation of RNA synthesis. A short synthetic RNA (5� AAAAAAAAGC 3�) was usedas a template in the de novo initiation assay. GTP and radiolabeled CTP were used as the initiating and the elongating nucleotides, respectively.A standard reaction mixture in a total volume of 20 �l contained 50 mM HEPES (pH 7.5), 10 mM �-mercaptoethanol (�-ME), 10 mM MgCl2,5 �M RNA template, 100 �M GTP, 100 �M CTP–10 �Ci of [�-33P]CTP, 1 �M HCV NS5B, and increasing concentrations (0 to 300 �M) of theanalogue compounds. The reaction mixture was incubated at 30°C for 30 min, and the RNA products were resolved on a 25% polyacrylamide gelelectrophoresis–6 M urea–Tris-borate-EDTA gel prior to PhosphorImaging analysis. Compounds I and II are shown in lanes 2 to 6 and 7 to 11,respectively. The labeled dinucleotide product (pppGpC) is indicated on the left.

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Synthesis of dinucleotide compounds. We initiated thisstudy by synthesizing two dinucleotide analogues (I and II)(Fig. 1) that contained a guanine base at the 5� end and a cy-tosine base at the 3� end. Modifications were introduced in thesugar moieties (2� or 3�-O-methyl) and the linker (phosphoro-thioate) for improvement of compound stability. In addition, a5�-phosphate group was added to further mimic the naturaldinucleotides. These compounds were guanine-cytosine an-alogues (pGpsC).

The dinucleotide compounds were synthesized on solid sup-port at a 10-�mol scale with an Applied Biosystems 394 DNA/RNA synthesizer. For dinucleotide I, 3�-dC-CPG 500 (GlenResearch) (compound 1 in Fig. 1A) was used as the startingmaterial. For dinucleotide II, 5�-O-dimethoxytrityl-N4-benzoyl-3�-O-methylcytidine-2�-O-[(2-cyanoethyl)-(N,N-diisopopyl)]phosphoramidite (compound 7 in Fig. 1B) was linked to uni-versal solid support. The synthesis of the dinucleotide com-pounds was carried out with the following common steps (Fig.1): (i) detritylation (3% dichloroacetic acid [DCA] in CH2Cl2for 3 min); (ii) coupling with the next monomer, 5�-O-dimethoxytrityl-N-isobutyryl-2�-O-methylguanosine-3�-O-[(2-cya-

noethyl)-(N,N-diisopopyl)]phosphoramidite (compound 2 in Fig.1A), using tetrazole as the coupling agent; (iii) oxidation usingBeaucage reagent to the phosphorothioate; (iv) detritylation; (v)coupling of the 5�-phosphate group using chemical phosphoryla-tion reagent 4 (compound 4 in Fig. 1A) (Glen Research); and (vi)oxidation using iodine-water to 5�-phosphate.

The chemical phosphorylation reagent used for addition ofthe 5�-phosphate had a terminal dimethoxytrityl (DMTr)group, which was utilized to facilitate reverse-phase high-per-formance liquid chromatography (HPLC) purification. Aftercompletion of the synthesis, dinucleotide I was treated withNH4OH at 60°C for 22 h, while dinucleotide II was treated at80°C for 8 h. This NH4OH treatment cleaved dinucleotidesfrom solid support and removed protecting groups from nucleo-bases and internucleoside phosphate linkage. The compoundswere then purified with C18 columns (Waters). The dilutetrifluoroacetic acid solution was used as a mobile phase toremove the DMTr protection group. The purified dinucleotidecompounds were characterized by mass spectrum (I, M�H� �683.62; II, M�H� � 713.42). HPLC purity was more than85%, with the single largest impurity not more than 3%.

Dinucleotide analogues inhibited de novo initiation of RNAsynthesis directed by HCV NS5B. To determine whether thesynthesized dinucleotide compounds (pGpsC analogues) caninhibit initiation of RNA replication by HCV NS5B, a de novoinitiation assay was performed, which used a small synthetic10-mer RNA (5� AAAAAAAAGC 3�) as the template. In thisassay, GTP (at 100 �M) was used as the initiating nucleotide,and formation of the dinucleotide product pppGpC was mon-itored by inclusion of [�-33P]CTP as the elongating nucleotidein the reaction. The 3�-terminal bases (GC-3�) of the templateRNA were chosen based on the following considerations. (i)

FIG. 3. Requirement of the 5�-phosphate group in the dinucleotidecompound for inhibitory activity. Inhibitory effects on de novo initia-tion of RNA synthesis by dinucleotide analogues I (lanes 2 and 3), II(lanes 4 and 5), and III (lanes 6 and 7) were compared (50 and 200�M). A short synthetic RNA (5� AAAAAAAAGC 3�) was used as thetemplate in the de novo initiation assay. GTP and radiolabeled CTPwere used as the initiating and elongating nucleotides, respectively (seethe legend to Fig. 2 for assay details). The structure of compound IIIis shown on the right.

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The 3�-terminal sequence of negative-strand HCV RNA(�GC 3�) serves as a template for multiple rounds of positive-strand RNA synthesis. (ii) The dinucleotide compounds syn-thesized mimick guanine-cytosine analogues. It has beendemonstrated previously that GTP is an efficient initiationmolecule for HCV NS5B in a number of assays (14, 19, 25, 28).As shown in Fig. 2, both dinucleotide analogues I (lanes 2 to 6)and II (lanes 7 to 11) had an inhibitory effect on NS5B-directedde novo initiation, with 50% inhibitory concentrations (IC50s)of 20 and 65 �M, respectively. This result indicated that thedinucleotide compounds were able to efficiently compete withthe initiating nucleotide GTP for binding to the polymeraseprotein and were able to inhibit the initiation of RNA syn-thesis.

The difference between these two dinucleotide analogueswas that one contains a 3�-O-methyl group at the C3�of thesecond ribose moiety (II), while the other contains a hydrogen

(3�-H) at the same position (I). The relatively weaker activityfor analogue II (higher IC50) implied that addition of a largergroup at the 3� position reduced the binding affinity to theNS5B polymerase and thus competed less effectively with theinitiating GTP

The 5�-phosphate group was important for the inhibitoryactivity of the dinucleotide analogues. Both analogues tested inFig. 2 contained a phosphate group at the 5� terminus. This wasbased on the assumption that the phosphate group might con-tribute to the binding affinity with NS5B polymerase. To dem-onstrate that such a phosphate group was indeed important forthe inhibitory activity of the dinucleotide compounds, a thirdanalogue (III) was tested in the same replication initiationassay. Analogue III possessed a similar structure to analoguesI and II, except the 5�-phosphate group was deleted. As shownin Fig. 3, analogue III lost its inhibitory activity on de novoinitiation of RNA synthesis, with an IC50 of �200 �M (com-

FIG. 4. Base pairing between the dinucleotide analogue and the template 3�-terminal bases is important for optimal activity. De novo initiationof RNA synthesis was tested with two synthetic RNA templates, which differ only in the terminal base (U versus C), in the presence of increasingconcentrations of dinucleotide analogue I (see Fig. 2 for structure). ATP and GTP were used as the initiating nucleotides, respectively, based onthe terminal base of the template RNA (ATP for 5� AAAAAAAAGU 3� and GTP for 5� AAAAAAAAGC 3�).

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pare lanes 6 and 7 with lanes 2 to 5). This result suggested thatthe 5�-phosphate group of the dinucleotide analogues playedan important role in the interaction with NS5B protein, thelack of which resulted in loss of binding affinity required forefficient competition with the initiating GTP.

Optimal inhibitory activity of the dinucleotide analogueswas dependent on the base-pairing potential between the an-alogues and the template 3�-terminal bases. To determinewhether the inhibition of de novo initiation by dinucleotideanalogues was template sequence specific and whether base-pairing potential between the compounds and the templateRNA played a role in stabilizing the compound-NS5B interac-tion, de novo initiation assays were performed with two sepa-rate RNA templates that contained different 3�-terminal bases(Fig. 4). One template (5� AAAAAAAAGU 3�) used ATP asthe initiating nucleotide, whereas the other (5� AAAAAAAAGC 3�) used GTP as the initiating nucleotide. The radiolabeleddinucleotide products generated from the de novo initiationassays were pppApC and pppGpC, respectively. The inhibitoryeffects of analogue I (which showed the highest activity inprevious assays) on RNA initiation from the two RNA tem-plates were compared. As shown in Fig. 4, this compoundshowed a more profound effect on GTP-initiated RNA synthe-sis (IC50 of 20 �M) than on ATP-initiated RNA synthesis(IC50 of 90 �M). This result, together with the fact that thedinucleotide compound tested was a guanine-cytosine (GC)analogue, suggested that the base-pairing potential between

the compound and the 3�-terminal bases of the template RNAplayed a positive role in maximizing the inhibitory activity onde novo initiation of RNA synthesis.

Inhibition of RNA synthesis initiated by dinucleotide prim-ers. Prior assays in determining the compounds’ activity wereperformed using mononucleotide (GTP or ATP) as the initi-ating molecule. We have shown previously that dinucleotideswere more efficient as initiating molecules than mononucle-otide in NS5B-directed RNA replication assays (27). It wouldbe interesting to determine whether the dinucleotide ana-logues were capable of inhibiting RNA synthesis initiated fromdinucleotide primers. To this end, radiolabeled dinucleotideprimer ([33P]GpC) was used to prime RNA synthesis (UTPincorporations) from the template RNA 5� AAAAAAAAGC3� in the presence of increasing concentrations of compound I.As shown in Fig. 5, the dinucleotide analogue was also active ininhibiting pGpC-initiated RNA synthesis (multiple rounds ofUMP incorporations), with an IC50 of approximately 50 �M(lanes 1 to 6). This result confirmed that the dinucleotideinhibitors were capable of competing with dinucleotide initiat-ing molecules and inhibiting RNA replication. The apparentreduction in the potency of the compound likely reflected thetighter binding between the enzyme and dinucleotide initiat-ing molecule, resulting in less efficient competition by thecompound.

Initiation of RNA synthesis by viral replication machineryrepresents a key step in the replication cycle of RNA viruses.It is generally believed that initiation is a rate-limiting step inviral RNA replication and that therapeutic interference at thisstep will effectively reduce the level of virus replication. So far,compounds specifically targeting the initiation process of viralRNA replication have not been thoroughly documented. Inthis study, we showed that dinucleotide compounds could serveas potential inhibitors of de novo initiation of RNA synthesis

FIG. 5. Inhibition of RNA synthesis initiated from a dinucleotideprimer (pGpC). The initiation assay reaction mixture contained, inaddition to the standard components listed in the legend to Fig. 2, 5�M template RNA (5� AAAAAAAAGC 3�), 5 �M end-labeled[33P]GpC primer, 100 �M UTP, and 1 �M NS5B enzyme. ExtendedRNA products were detected from extension of the labeled primer dueto multiple UMP incorporations.

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directed by HCV NS5B polymerase. This observation was inaccordance with our previous results showing that dinucleo-tides were efficient initiating molecules for HCV NS5B-di-rected RNA replication. The use of dinucleotide analogues asinhibitors to target the initiation step of viral RNA synthesisrepresents a novel approach to antiviral interference.

Despite the successful confirmation of the concept that dinu-cleotide analogues are effective inhibitors of de novo RNAreplication, significant challenges remain before further devel-opment of this class of compounds for direct therapeutic ap-plication can be considered. Dinucleotide molecules, especiallyRNA, are generally unstable. The intramolecular nucleophilicattack by the 2�-hydroxyl group in the sugar moiety on the ad-jacent phosphate bond causes the degradation of the dinucleo-tides to mononucleotides and even further to nucleosides.Dinucleotides can also be hydrolyzed by water and other nu-cleophiles present in the biological system. In addition, the5�-phosphate dinucleotides, which were shown to be more ef-fective in inhibiting de novo initiation of RNA replication, areeven less stable because of the presence of the additionalphosphate group. Furthermore, the polyanionic nature of thedinucleotide molecules strongly hampers the penetration ofsuch compounds through cell membrane due to the high lipo-philicity on the membrane surface. Finally, dinucleotide mol-ecules can also bind to other nucleic acids through hydrogenbonds, which may result in a loss of selectivity for a predeter-mined target in vivo.

The pGpsC type of dinucleotide analogues used in this studycontained a phosphorothioate linker. Such linker is believed toenhance the stability of the dinucleotide compounds over thenatural nucleic acid. For the same purpose, the 2�-hydroxylgroup of the sugar moiety was further modified by methylationso that the 2�-hydroxyl group is not available for intramolecularhydrolysis. Nucleic acids with 2�-O-methyl modifications havebeen reported to possess improved stability, lipophilicity, andbioavailability, as well as other pharmacological properties.The physicochemical and pharmacological features of thedinucleotide compounds can be further improved by modi-fications of the phosphate groups in reducing their anioniccharacter and, in the mean time, increasing lipophilicity. Inthis case, the various conjugation strategies used for improv-ing properties of oligonucleotide compounds may be ap-plied.

Despite all of these challenges, the demonstration that dinu-cleotide analogues can effectively inhibit de novo initiation ofRNA replication by HCV NS5B confirms the proof of princi-ple for this novel antiviral approach. With future advance-ments in addressing the outstanding issues related to the dinu-cleotide compounds, this class of compounds may eventuallyreach a stage of development for therapy against importantviral diseases.

We thank Wayne Cheney, Vicky Lai, Nigel Horscroft, Jim Wu, JaeHoon Shim, Todd Appleby, and Nanhua Yao for helpful discussions.

This work was supported by Ribapharm, Inc.

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